A multi-faceted research project is directed aimed at computational studies of enzymatic processes in aqueous solution. The theoretical approach centers on molecular dynamics free energy simulations of enzymes, making use of combined quantum mechanical and molecular mechanical (QM/MM) methods. A major goal is to increase the capability of QM/MM methods and to achieve greater accuracy than conventional approaches. We propose to further improve the mixed molecular orbital and valence bond (MOVB) theory, coupled with the self-consistent charge tight-bonding density functional theory (SCC-DFTB) and extension to the CHARMM program with ab initio and DFT methods, such that the theoretical model can be conveniently calibrated, tested and used by experimental biochemists as a research tool to help interpret experiment findings. The MOVB method has been developed at theoretical levels that include ab initio and semiempirical molecular orbital and density functional theory. One goal of the present study is to incorporate the procedure into molecular dynamics simulation programs for effectively modeling enzymatic reactions. A major thrust of this project is to provide a deeper understanding of the underlying principles and mechanisms of enzymatic reactions. During this grant period, we focus on the catalytic mechanism of histone lysine demethylases with emphasis on the Jumonji C domain containing enzymes, which belong to a large class of enzymes that utilize a non-heme high- valent iron-oxo intermediate. Histone lysine demethylases along with other histone protein modifying enzymes play a critical role in epigenetic regulation and have been found to be associated with cancer development and progress. In addition, we seek to address the general properties of enzymatic proton-coupled electron transfer reactions and the effects of protein dynamics and enzyme reorganization energies on these processes. The MOVB method provides an important research tool to study these questions, and the results will be of general importance to protein engineering and inhibitor design. PHS 398/2590 (Rev. 06/09) Page Continuation Format Page PUBLIC HEALTH RELEVANCE: Proteins are workhorses in the living cell, performing all the fundamental tasks from metabolism to cell growth. An important goal is to develop pharmaceutical drugs against protein targets that are responsible for cancer growth and other diseases. The research described in this proposal aims at the fundamental understanding of the mechanism and function of enzymes, proteins that catalyze chemical reactions and bioenergy transformation, and the knowledge gained from these studies can help design inhibitors and engineer specialized proteins for biomedical and industrial applications. PHS 398/2590 (Rev. 06/09) Page Continuation Format Page